We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5 end of the chicken beta-globin locus, which is capable of blocking enhancer- promoter interaction when it lies between them. We had earlier narrowed down this activity to a small DNA fragment within the 1.2 kb sequence. We purified the protein responsible for this activity and identified it as CTCF, which is a known factor ubiquitous in vertebrates, with both stimulatory and inhibitory effects on expression of a variety of genes. We have shown that CTCF sites are present in other vertebrate gene loci, at sites reported by others to have enhancer blocking activity. In parallel work we have examined the organization of the 3 end of the chicken beta globin locus, and identified by changes in nuclease sensitivity the 3 boundary of the locus. We find at this boundary a constitutive hypersensitive site very similar to the one that marks the 5 boundary of the locus. It has enhancer blocking properties like those of the upstream element and contains a CTCF site. Beyond this we find a complete open reading frame for an odorantreceptor (OR). In simultaneous work (in collaboration with the laboratories of Mark Groudine, Fred Hutchinson Cancer Research Center and Richard Axel, Columbia Univ.) we identified members of the same OR family near the mouse beta globin locus. The chicken OR is not expressed in erythroid cells, raising the possibility that the 3 boundary element serves to prevent interaction between the two gene systems, just as the 5 boundary may prevent interaction of the globin locus with the folate receptor gene that we have recently located only 16 kb 5 of the globin locus. We have also continued studies of the mechanism by which RNA polymerase is able to transcribe chromatin templates. In earlier work we used single nucleosomes as templates and showed that the histone octamer was capable of transferring out of the path of the polymerase without letting go of the DNA. We proposed a model for the intermediates in this process. We have now obtained in collaboration with Chris Woodcock (Univ. Massachusetts) electron cryo- micrographs of these intermediates, which confirm the solution observations, and provide more detailed information about their structure. - chromatin structure, globin gene, erythroid cells, insulator, enhancer blocking, folate receptor, olfactory receptor